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Forensic Biology: Screening Standard Operating Procedures
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1. This procedure can be used when larger items of evidence are presented FAD BIO SOP 7 1 Page 3 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 7 Semen Detection 1 Spread the clothing item bedding towel or other item flat onto a non porous surface use clips if necessary 2 Moisten filter paper with sterile diH2O Lay the moist filter paper over the item or area of the item to be tested Press the paper firmly against the item for approximately 30 seconds to 5 minutes to allow for any possible acid phosphatase to transfer to the paper Mark the position of the paper on the item Remove the filter paper Spray or apply drop s of AP test reagent to the filter paper Observe the treated filter paper for up to 60 seconds for a color change After interpretation mark the tested areas with results Discard used filter paper by placing it in the biohazard trash 9 Oo Na EA Interpretation Should a color reaction take place within 60 seconds the result only indicates the possible presence of semen the test is therefore a presumptive test Grade the reaction according to the time it takes for the color to appear as follows e 0 15 seconds 4 e 16 30 seconds 3 e 31 45 seconds 24 e 46 60 seconds 14 e Nochange or color change after more than 60 seconds negative A negative result indicates acid phosphatase is absent or below the de
2. Forensic Biology Screening Standard Operating Procedures Forensic Analysis Division Standard Operating Procedures Biology Table of Contents Table of Contents 1 Overview 2 Facilities 2 1 Work Areas 2 2 Contamination 2 3 Safety 3 Equipment Quality Control and Maintenance 3 1 Maintenance and Calibration 3 2 Equipment 4 Quality Assurance 4 1 Chemical and Reagent Labels 4 2 Quality Control Checks 4 3 Logbook 4 4 Proficiency Testing 5 Evidence Evaluation and Handling 5 1 Case Acceptance and Evaluation 5 2 Evidence Handling 6 Blood Detection 6 1 Phenolphthalein Test Kit 6 2 Luminol 6 3 ABAcard HemaTrace Test Devices 6 4 References 7 Semen Detection 7 1 Alternate Light Source 7 2 Acid Phosphatase Spot Test 7 3 Body Fluid Extraction 7 4 Microscopic Spermatozoa Examination 7 5 SERATEC PSA p30 Semiquant Test 7 6 Guidelines for Sexual Assault Cases When Contact or Digital Penetration is Alleged 7 7 References 8 Trace Observation Collection 9 Contact DNA Collection 10 Portioning for DNA Analysis 11 Case Records 11 1 Examination Documentation 11 2 Reports 11 3 Examination Counts 12 Abbreviations 13 Chelex Extraction of DNA 14 Plexor HY Quantification FAD BIO SOP TOC 1 Page 2 of 2 Revision Date May 2 2014 Effective Date June 1 2014 Issued by Technical Leader Forensic Biology Screening Overview Forensic Analysis Division Standard Operating Procedures Biology 1 Overview O
3. Test Devices Scope ABAcard HematTrace test kits are used to determine the presence of human blood in suspected blood stains Instructions for use and interpretation are in the ABAcard HematTrace procedure Safety Wear gloves lab coat mask and other proper laboratory attire Broken skin should be covered Equipment and Supplies e ABAcard HematTrace test device s e ABAcard HemaTrace extraction buffer e Pipette and pipette tips e Known blood sample e Timer Each new lot of ABAcard HemaTrace test kits must be subjected to the appropriate in house quality control test as outlined below 1 Make a 1 10 dilution from a known human blood sample in extraction buffer provided in kit 2 Add 150ul of dilution to sample well S of one test device to be used as a positive control 3 Add 150 ul of extraction buffer from kit to sample well of second test device to be used as a negative control Read results up to 10 minutes from application of sample 5 The 1 10 dilution of known human blood must give a positive result The extraction buffer provided in the kit must give a negative result Storage Labeling and Expiration The test devices must be stored at room temperature below 82 F and must remain in the sealed pouch until use Label each test device with the name of the appropriate sample used The test kits may be used on casework samples up to the expiration date stated on the box 4 2 6 Phosphat
4. prior Click Next Fill in the Operator Name initials are acceptable a The remaining fields can be left blank FAD BIO SOP 14 1 Page 8 of 12 Issued by Technical Leader Oo Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification 7 Click Next 8 Click the upper Browse button to add the Amplification file 9 Navigate to the previously saved Delta Rn file i e AB092012 delta 10 Select file and click open 11 Click the lower Browse button to add the Melt file 12 Navigate to the previously saved Raw amp Derivative data file i e AB092012 diss 13 Select file and click open 14 Under the Advanced Options a Click Edit under Analysis Template b Select Defaults gt Reset to Default c Click OK ile Import To import your Applied Biosystems 7500 run use the file dialog below to specify the Delta Rn Ampimication and Dissociatian Mel files you have exported Pengiicatan Filename Cancel lt Back 15 Click Finish 16 A window will appear with the run data Data Analysis and Report Generation 1 Make sure the FL Autosomal Tab is selected 2 With all samples selected go to Edit gt Add Standard Curve a Alternatively you can hit the d key Caps lock cannot be on 3 Select the CO560 Y Tab 4 With all samples selected go to Edit gt Add Standard Curve a Alternatively you can hit the d key Caps lock cannot be on 5
5. provided in the trace rooms for analysis Lab coats used in other areas of the laboratory will not be taken into the trace rooms due to possible contamination For any method used tables and tools will be cleaned with bleach solution or other lab approved cleaning solution before each use to remove any extraneous debris At a minimum tools will be cleaned and new bench paper will be used for each item when multiple items are received packaged together Generally for any method used a clean sheet of butcher paper will be used on the table for each evidence package opened Evidence from victim s and suspect s will be examined in separate rooms to prevent cross contamination The examiner will change lab coats between the examination of items from victim s and suspect s If separate rooms are not available items from victim s and suspect s can be processed in the same room after a period of 7 days 8 2 Procedure Equipment Materials and Reagents e Butcher paper e Tweezers e Plastic zipper bags e Clear packing tape or trace evidence lifters There are several options available for ensuring that trace evidence is properly collected stored preserved These are as follows 1 Visual examination locate and remove trace evidence from the evidence item with tweezers Recovered trace will be placed in a suitable labeled container zipper bags or on tape to prevent loss or contamination of the sample FAD BIO SOP 8 1 Page 2 of 3 Issued b
6. retain External swabs without clear notes on why it was collected outer envelope only states Breast swabs e Press out do not retain negatives Intimate swabs Penile and other penetration e Within 24 hours snippet test retain regardless of semen results e Beyond 24 hours press out do not retain negatives Non penile penetration e Within 24 hours retain e Beyond 24 hours NTC Incompetent ex mentally challenged infants children e Snippet test retain regardless of semen results Unconscious ex doesn t know what happened drunk drugged e External swabs o Bathed NTC add bathed statement in report o Not bathed retain e Intimate swabs o Press out do not retain negatives FAD BIO SOP 7 1 Page 9 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 7 Semen Detection o Unless specifically states a digital contact situation o Within 24 hours snippet test retain regardless of semen results o Beyond 24 hours press out do not retain negatives Any information indicating that semen may be present should always be considered and samples processed accordingly regardless of above guidelines ex he ejaculated on my neck shorts etc 7 7 References 1 http www microptics com eng products sperm_ analysis sca morphology html Accessed July 21 2012 http www historyforkids org scienceforkids biology cells transportati
7. 1 2014 Forensic Biology Screening Facilities Forensic Analysis Division Standard Operating Procedures Biology 2 Facilities 2 Facilities 2 Facilities The Biology section will be designed to provide adeguate space and setup to perform casework analysis General facility reguirements are described in the Guality Manual 21 Work Areas The Biology section will have space for evidence examination The tasks performed in this area can include blood and semen fluid identification swab collections cutting of stains portioning of swabs and cuttings microscopy and trace evidence collection 2 2 Contamination With the facility provided setup and procedures will be designed to minimize the potential for DNA contamination Traffic in and through areas in which testing occurs will be minimized Prevention and Decontamination 10 bleach or DNA Away where bleach may be harmful to instruments or equipment will be used to decontaminate utensils and workbenches Other commercial decontaminants may be used only if they have been demonstrated to destroy or inactivate DNA Wear disposable gloves during all testing and reagent preparation Change gloves freguently and whenever gloves may have become contaminated Discard gloves when leaving a work area Use sterile disposable pipette tips and microcentrifuge tubes Change pipette tips between samples Centrifuge all liquid to the bottom of closed microcentrifuge tubes before openin
8. 5um FAD BIO SOP 7 1 Page 5 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 7 Semen Detection Identifying characteristics for spermatozoa PON 5 Size head is approximately 4 6 um x 2 6 um x 1 5um Morphology oval to round or teardrop shaped structure Acrosomal cap at tip of sperm unstained or lightly stained Staining dark pink to red head with unstained or lightly stained acrosomal cap tails green or blue if present Presence of a flagella or tail may not be present EQUIPMENT MATERIALS AND REAGENTS Microscope slide Body fluid extract Pipette and pipette tips Heat block Christmas Tree Stain Solution A and Solution B Timer Tap water 95 ethanol Compound microscope Immersion oil Procedure If slide has already been prepared proceed to step 4 1 2 Pipette 10 ul of the supernatant from the body fluid extract onto a glass slide Fix smear by heating the slide on the heat block for approximately 5 minutes Stain the slide a Cover the sample area on the slide with Solution A for 10 minutes b Wash with tap water by gentle flooding c Cover the sample area with Solution B for 15 seconds d Wash with tap water by gentle flooding FAD BIO SOP 7 1 Page 6 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 7 Semen D
9. Biology Screening Portioning for DNA Analysis Forensic Analysis Division Standard Operating Procedures Biology 10 Portioning for DNA Analysis 10 Portioning for DNA Analysis 10 1 Scope After serological analysis any evidence that will be retained in the laboratory can be portioned for potential DNA analysis Please refer to SOP 5 Evidence Evaluation and Handling for guidelines on general evidence handling and the consumption of evidence in particular These should be treated as guidelines only good judgment and case specifics may warrant a deviation from what is prescribed below 10 2 Eguipment Materials and Reagents e Scalpel or scissors e Microcentrifuge tube s e Weighing paper or butcher paper 10 3 Procedure NOTE Make every effort to preserve at least half of the sample for re analysis If this is unavoidable written permission must be obtained 1 Set the evidence onto paper 2 Portion the evidence according to the chart below Swabs P30 only no spermatozoa Ya of total swab s lt 10 spermatozoa Ye of total swab s 2 10 spermatozoa Remainder of swab cut for p30 or micro HemaTrace Ye remainder of swab cut for HematTrace QNS Va of total swab s Contact Ye of total swab s Bloodstain card 0 5 cm from center of stain Known saliva swabs Y2 of 1 swab FAD BIO SOP 10 1 Page 2 of 3 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Sta
10. In the case of luminol the catalytic oxidation of the substrate compound produces light Because the reagents are applied as a spray over a large area the test is primarily used in the visualization of bloodstain patterns As with similar presumptive blood tests a number of oxidizing substances will produce a false positive reaction Should a positive reaction take place the result only suggests the presence of blood the test is therefore a presumptive test Safety This reagent presents the following hazards luminol is an irritant Sodium perborate and sodium carbonate are toxic and irritating Avoid breathing dust do not get in eyes on skin or on clothing Avoid breathing sprayed solution Wash hands after handling Gloves must be worn during testing Broken skin should be covered Equipment and Supplies e Luminol powder 3 aminophthalhydrazide e Sodium carbonate e Sodium perborate e Sterile diH2O e Two darkened or foil wrapped spray bottles e Scale Preparation Reagent A 1 Measure 250 mL of sterile diH O 2 Add 2 5 g of sodium carbonate 3 Mix until all of the powder is dissolved FAD BIO SOP 6 1 Page 3 of 6 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 6 Blood Detection 4 Add 0 5 g of luminol powder 5 Mix until all of the powder is dissolved Preparation Reagent B 1 Measure 250 mL of sterile diH O 2 Add 6 5 g of sodium perborat
11. Select the Sample Names Tab a Open the Excel Plexor HY worksheet saved previously b Select and Copy the wells that were used for the run c Return to the Plexor Analysis software 6 Select Edit gt Paste Sample Names from Template a Alternatively you can hit the Ctrl t key 7 Click Accept Changes in the bottom right of the window FAD BIO SOP 14 1 Page 9 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification 8 Click on the Standard Curves Tab a Evaluate the standard curves as follows i Click on the FL Autosomal tab 1 The Autosomal curve the slope must be between 3 1 to 3 7 and R2 2 0 990 ii Click on the CO560 Y tab 1 The Y curve the slope must be between 3 0 to 3 6 and R2 2 0 990 NOTE A limited number of outlier points may be omitted from the curve to bring these values within acceptable range If these values are not within range the procedure must be re run Removal of more than 3 data points must be approved by the Technical Leader Points can be removed by changing the sample type under the PCR Curves tab Refer to Plexor HY manual for more detail 9 Select Forensics gt Set Normalization and IPC parameters a Do not change any settings 10 Click Ok on the window that pops up a Give the software a minute and the window will change to the Forensic report tab under the Reports tab 11 Check
12. The water should be clear and clean with no evidence of bacterial fungal growth or rust The following procedure should be performed as needed Wash the inside of the ultrasonic cleaner with detergent and rinse well with water Fill bath with the appropriate quantity of diH2O water Mini centrifuges Mini centrifuges are bench top unrefrigerated centrifuges that have been designed for quick spin downs from tube walls and caps of tubes These mini centrifuges are equipped with a circular one sized rotor The maximum speed is 6000 rpm The relative centrifugal force can be determined as outlined in the manufacturer s instructions if required FAD BIO SOP 3 1 Page 2 of 8 Issued by Technical Leader Revision Date May 30 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 3 Equipment Quality Control and Maintenance Please consult the appropriate eguipment manual for specific instructions on maintenance and operation of the mini centrifuge These centrifuges will primarily be used for removing liquid from the cap of a microcentrifuge tube They will not be used for pelleting cellular material Thermometers All thermometers must be either calibrated or their accuracy must be verified to ensure the temperature is being accurately measured NIST traceable thermometers must be purchased or re certified at least once every two years A NIST traceable thermometer certified for two years and used for conducting performance
13. and after each use the box should be wiped inside and out with a 10 bleach solution or other lab approved decontaminant and dried with a soft cloth Do not autoclave immerse or abrade the unit Refrigerators and Freezers Please refer the Laboratory Quality Manual for refrigerator and freezer maintenance Reagents and evidence will be stored separately either in separate cooling units or in separate space within those units Pipettes Volumetric pipettes are used to accurately and precisely measure the volume of a solution to be delivered The accuracy of the measurement is dependent upon the correct technigue of the user as well as the proper maintenance and performance of the pipette The pipette should be able to provide accuracy precision repeatability and reproducibility over the full range of the pipette s capabilities The Biology section is supplied with pipettes that cover the volume range from 0 1 1000 ul Calibration will be performed at least annually on these instruments When a pipette is determined to be performing improperly it will be returned to the manufacturer or another gualified repair technician so that the problem may be identified and corrected Guality control data should include the type of pipette volume range model and serial number Procedure 1 Set the volume of the pipette using the knob in the pipetting push button 2 Firmly attach the appropriate tip per manufacturer s recommendations to the pipett
14. be included in the report as to requesting trace analysis if needed Note if any items listed were not analyzed It may be appropriate to include in the report a request for the submission of known samples from any of the following suspects complainants witnesses consensual sex partners and or other individuals for the purposes of elimination Although interpretation of individual tests are discussed in the appropriate protocols analysis results must be considered together to ensure that conclusions take into account all reasonable possibilities During analysis the examiner must continuously monitor results for problems and inconsistencies that may ultimately affect final conclusions Therefore careful review of notes and results by the examiner should be undertaken before conclusions are drawn A supervisor or qualified examiner may assist in the developing of the appropriate conclusions Slightly different situations may lead to different conclusions When in doubt consult with other qualified examiners and or the supervisor Reports will include the disposition of all items of evidence whether they are retained within the laboratory or returned to the submitting agency 11 3 Examination Counts Information related to the number of examinations performed during each analysis can be documented on the Serology Examination Count Sheet in LIMS This information can be used by laboratory management to accurately capture metrics such a
15. calibration records and repair activities shall be maintained and recorded in an equipment calibration and maintenance log This log will include at a minimum the date activity laboratory personnel performing or overseeing the activity non crime laboratory technician s performing or overseeing the activity and a record of quality control checks performed to verify operation prior to returning a piece of equipment to casework use The section supervisor and QA Manager are responsible for ensuring all systems are checked and maintained as required Whenever practical equipment that requires calibration will be labeled with the date when last calibrated and the date or expiration criteria when recalibration is due Critical equipment is any piece of equipment that must be maintained in a proper working order to ensure the reliability of results produced Some critical equipment and associated maintenance requirements can be found in the Quality Manual Additional equipment and maintenance requirements are included here Equipment Operation Manuals Equipment operation manuals will be readily available to each examiner approved to use the equipment 3 2 Equipment Ultrasonic Cleaner Ultrasonic cleaners use ultrasonic energy in the form of sound waves to create a mechanical scrubbing action to loosen debris from all surfaces that the solution touches This equipment s condition is routinely maintained for the Biology section s procedures
16. checks on equipment shall require the annual performance check A NIST traceable thermometer certified for two years that is not used for conducting performance checks does not require the annual performance checks and may be used until the certification expires A NIST traceable thermometer to be used beyond its certification date shall be recertified or be subject to the annual performance check requirements The minimum requirements of a performance check of a thermometer used for performing performance checks may be accomplished 1 through certification by an outside vendor or 2 in house by the comparison of one or more temperature readings at various time intervals against another NIST traceable thermometer Thermometers that are not NIST traceable must be verified annually by comparison against a NIST traceable thermometer Any deviation from the NIST traceable thermometer will be noted and all other readings adjusted by the same amount and in the same direction for each thermometer If the deviation is greater than 3 C the thermometer should be serviced or replaced Thermometers should be handled carefully to avoid breakage Thermometers may be wiped clean to facilitate easy and accurate readings If any discontinuities are detected repair or replace the thermometer Autoclaves Autoclaves are used to sterilize solutions glassware and instruments by subjecting them to high pressure and high heat simultaneously Autoclaves also may b
17. in 10 minutes The sample is added to a sample well where any detectable human hemoglobin hHb present in the sample will bind with mobile monoclonal antihuman Hb antibody which has an attached pink dye particle The resultant mobile antigen antibody complex migrates through an absorbent strip to an area where an immobile polyclonal antihuman Hb antibody is bound The mobile antigen antibody complex binds to the immobile antibody creating an antibody antigen antibody sandwich Conjugated pink dye particles become visible in the area of immobilized antibody in the test area T when the hHb concentration in the sample exceeds 0 05 ug ml The resultant FAD BIO SOP 6 1 Page 4 of 6 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 6 Blood Detection pink band indicates a positive result As an internal positive control hHb antibody dye conjugates cannot bind to the antibody in the test area T but are captured by an immobilized anti immunoglobulin antibody present in the control area C forming a complex The captured pink dye particles will thus form a band in the control area C indicating that the test has worked properly and that appropriate procedures have been followed Hemoglobin from the Family Mustelidae ferrets and higher primates also gives a positive result The examiner must evaluate the likelihood of blood from either of these sources be
18. now show an X through them 4 On the menu bar select Analysis gt Analyze a Alternatively the green arrow in the menu can be clicked 17500 System SDS Software Duo samples sds Absolute Quantification 5 File View Took Instrument analsis Window Help is ERE MED m 5 Exporting Files a Select File gt Export gt Delta Rn b Save the file with the run name and the suffix delta i e AB092012 delta i Save the file to the Desktop with the run file c Select File gt Export gt Dissociation gt Raw amp Derivative data d Save the file with the run name and the suffix diss i e AB092012 diss i Save the file to the Desktop with the run file e Do NOT open the export files This will corrupt them for later use 6 Close the 7500 run file and 7500 SDS software a Click Yes to Save if prompted to do so 7 Move the three saved files to the appropriate month and year folder of travfs0 1 CrimeLab SHARED Biologylnstruments 7500PCR Plexor HY 8 Remove and discard the plate from the 7500 9 Turn off the 7500 Plexor HY Analysis software Note The following instructions are suitable on software that has been appropriately initialized Software set up and file import 1 Start the Plexor Analysis Software 2 Select File gt Import New Run 3 Fill in the Assay Name with the same name as the 7500 run file i e AB092012 4 Make sure that the instrument is set to Applied Biosystems 7500 SDS v1 4 amp
19. of Solution A Procedure 1 Moisten a sterile cotton swab or piece of filter paper with sterile diH O 2 Rub or press down on the suspected bloodstain with a sterile cotton swab or filter paper to allow for any possible blood to transfer 3 Apply approximately 1 drop of Solution B to the swab or filter paper FAD BIO SOP 6 1 Page 2 of 6 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 6 Blood Detection 4 Observe briefly to identify color change A green color is often observed when blood is present any other color change should be documented 5 Add approximately 1 drop of Solution C to the swab or filter paper 6 Observe the swab or filter paper for an immediate pink color change indicating a positive reaction 7 After interpretation mark the items areas tested with the results Discard the swab or filter paper by placing it in the biohazard trash Interpretation Appearance of an immediate pink color change after the application of Solution C is a presumptive positive result for blood No color change indicates a presumptive negative result for blood A negative result indicates that blood is absent or below the detection threshold Substrate controls may be included in body fluid identification tests as appropriate 6 2 Luminol Scope Catalytic tests for blood are based on the peroxidase like activity exhibited by the heme group of hemoglobin
20. of Trace Evidence However it is permissible for sample descriptions to include adjectives such as apparent or like when describing trace evidence e g hair like or apparent fibers 8 3 Reference Saferstein R 2007 Criminalistics An Introduction to Forensic Science Ninth Edition Upper Saddle River New Jersey Pearson Prentice Hall FAD BIO SOP 8 1 Page 3 of3 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Forensic Biology Screening Contact DNA Collection Forensic Analysis Division Standard Operating Procedures Biology 9 Contact DNA Collection 9 Contact DNA Collection 9 1 Scope This procedure may be used when necessary for the collection of contact DNA evidence prior to forensic biology analysis This laboratory does not currently screen for the presence of contact DNA through a method such as microscopic examination for the presence of epithelial cells Contact DNA is generally very limited in nature and consumption during screening may further reduce the potential success during DNA analysis For this reason when an item is suspected of containing contact DNA efforts to collect possible contact DNA will be made These samples may then be submitted for DNA analysis 9 2 Eguipment Materials and Reagents Sterile cotton swabs Sterile reagent grade water Swab drying box optional 9 3 Procedure 1 2 Moisten swab s with sterile diH O Swab the
21. of light directly from the unit can cause burns and other skin damage It is essential that proper eye protection be provided Avoid looking directly at the light source reflected or refracted light Remove all unnecessary reflective surfaces from exam room Wear goggles gloves lab coat and other proper laboratory attire Mini Crimescope MCS 400 ALS If the bulb fails suddenly stops emitting light it may leak vapors for afew minutes To be safe immediately leave the room and let the unit run fans for 5 10 minutes until vapors are vented Eguipment and Supplies e ALS set at 400 or 455 nm e Orange and or yellow glasses e Known semen sample e Known non semen sample Prior to use each day the ALS must be subjected to the appropriate in house quality control test as outlined below Turn on power switch and ensure the fans are working Wear the proper glasses Turn on lamp switch Darken the examination room Direct the ALS at the samples The known semen sample must fluoresce indicating a positive result BL DE ee eS The known non semen sample must not fluoresce indicating a negative result FAD BIO SOP 4 2 Page 4 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Guality Assurance When ready to shut down the ALS turn off lamp switch and wait for the unit to cool down 5 minutes Then turn off the power switch The resu
22. on case work documentation Non standard scientific abbreviations those not found in the dictionary and abbreviations not contained on this list must be defined on each examination document on which it is used Revision Date May 2 2014 Effective Date June 1 2014 none FNSC fingernail scrapings clippings PR HPD Property Room an approximately FTC found to contain PR payroll number ALS alternate light source HPS hospital patient sticker PREP preparation AP acid phosphatase HRS hours PT patient APP apparent HT HemaTrace Q questioned sample AR administrative review INC inconclusive QNS Pala i BC barcode INC Incident Number R B reddish brown BSC bloodstain card IPC pie ey Pesing or POR REP representative BUC buccal IT item REC D received e containing K known standard sample RMS E Management CAPT captain KBS known buccal swabs RPR return to property room COMM communication KSS Known saliva swabs S SEC second COMPL complainant LG large SA sexual assault CONT D continued LIMS son AN N S CELLS sperm cells CS Controlled Substances LT lieutenant SCRN Screening CSP Consensual sex partner MED medium SF sperm fraction CTRL control MICRO sperm search by microscopy SGT sergeant D depleted MFG manufacturer SM small DOB date of birth N A not applicable ST stain diH20 deionized water NEG negative STC said to contain DISP disposition NSO no
23. stains observed SUPPL supplement ECD excessive cellular debris NTC no testing conducted SUSP suspect na Southwestern Institute E CELLS epithelial cells NTO no trace observed SWIFS ot Forensic Sdien es EF epithelial fraction NSTO no significant trace observed TEMP temperature EMS Evidence Management System NVB no visible bloodstain TNTC too numerous to count ENV envelope OFTC opened found to contain TOX Toxicology EtOH ethanol OLO online offense system TR technical review EVID evidence PBS phosphate buffered saline UNK unknown EXP expiration PG PP page pages VAG vaginal F frozen PHT phenolphthalein W with FA Firearms PKGD PKG D packaged WIF walk in freezer FCN Forensic case number POS positive YOA years of age FNS fingernail scrapings POSS possible FAD BIO SOP 12 1 Page 2 of2 Issued by Technical Leader Forensic Biology Screening Chelex Extraction of DNA Forensic Analysis Division Standard Operating Procedures Biology 13 Chelex Extraction of DNA 13 Chelex Extraction of DNA This procedure will allow for the extraction of DNA from samples containing semen The Chelex extraction is an efficient and guick method to obtain the DNA from the samples The addition of DTT will allow for the digestion of samples that contain semen Samples may or may not contain semen Safety Body fluids tissues and extracts may contain infective agents Use universal precautions during evidence handling Follow instructions for reagent prepa
24. suspected area s on the evidence with the tip s of the cotton swab s Efforts to concentrate the possible contact DNA on the tip of the swab should be made This will hopefully serve to increase chances of DNA success not only by concentrating the possible DNA but also in the absence of visible staining later portioning will more likely capture DNA if the correct area of the swab is selected for portioning As the area to be swabbed increases in size so too may the number of swabs needed to adequately collect the evidence 3 The wet swab may be followed by a dry swab on non absorbent surfaces 4 Avoid any suspected semen or blood stains These sources of biological material generally contain more DNA and will potentially mask any lower level contact DNA These stains may be processed separately Allow swab s to completely dry in a swab drying box for 30 minutes Alternatively the swab s may be air dried overnight in a secure and clean environment Sometimes such as in the case of fingernail scrapings and clippings suspected bloodstains cannot always be avoided In such instances the analyst will look for any visible red brown staining and document accordingly prior to swabbing the item It may be appropriate to forgo blood or semen detection in order to maximize potential contact DNA for DNA anlaysis FAD BIO SOP 9 1 Page 2 of2 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Forensic
25. that the first location is A1 a Click on Location so the samples will be sorted by location 12 Select File gt Save Analysis File 13 Title the file with initials and run date i e AB092412 14 Save the file to the appropriate month and year folder of travfs01 CrimeLab SHARED Biologylnstruments 7500PCR Plexor HY Data interpretation 1 Check that the IPC Status column is OK for all samples NOTE A Check IPC value indicates the possible presence of inhibitors For any samples that say Check IPC select that sample and examine the PCR curves under the PCR Curves tab The absence of an IPC value under the CR610 IPC tab and a quantification value for a sample indicates either complete inhibition or a failure of the amplification reaction The sample may be rerun Alternatively a dilution may be made and run again with Plexor HY While the dilution would contain fewer inhibitors it would also contain less DNA and may not be a true negative 2 Check that the Curve Status column is OK for all samples a Depending on sample type standard non template control or sample three other options may be displayed depending on sample type i Check STD displayed for samples designated as standard controls ii Check NTC displayed for samples designated as non template controls FAD BIO SOP 14 1 Page 10 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Pr
26. the container with the date received and or date opened In general the manufacturer s labeling will be followed to determine expiration dates of purchased chemicals and reagents If no manufacturer expiration exists for a purchased reagent it will be considered expired 5 years from the date received and should be marked on the container Prepared reagent labels will include the reagent name initials of preparer and expiration date In general most solutions prepared in the Biology section shall expire 1 year from the date of preparation However the expiration date of the overall reagent will be no later than the expiration date of the individual reagent with the nearest expiration date unless otherwise specified Additional information may be documented in a reagent log The lot number contains the date prepared and the initials of the preparer e g mmddyyinitials 4 2 Quality Control Checks The following shall be tested to confirm that they are working properly with a positive and negative control each day used e Acid Phosphatase Spot Test e Alternate Light Source e Luminol mixture e Phenolphthalein test kit The following shall be quality control checked to confirm they are working properly with each new lot received e ABAcard HematTrace kits e Phosphate Buffered Saline 1X e SERI Christmas Tree Stain FAD BIO SOP 4 2 Page 2 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1
27. the slide a Cover the sample area on the slide with Solution A for 10 minutes Wash with tap water by gentle flooding Cover the sample area with Solution B for 15 seconds Wash with tap water by gentle flooding papp Flood the slide with 95 ethanol and allow the slide to dry for at least 5 minutes on the heat block Examine the slide for the presence of spermatozoa at 1000X magnification Sperm heads must be stained red and any observed tails must be stained green indicating a positive result Storage and Expiration The stain must be stored at room temperature The stain may be used on casework samples up to the expiration date stated on the bottles FAD BIO SOP 4 2 Page 10 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Guality Assurance 4 2 8 SERATEC PSA Semiguant Test Devices Scope SERATEC PSA Semiguant test devices are used to indicate the possible presence of p30 in suspected semen stains Instructions for use and interpretation are in SERATEC PSA Semiguant test procedure Safety Wear gloves lab coat mask and other proper laboratory attire Broken skin should be covered Eguipment and Supplies e SERATEC PSA Semiquant test device s e 1X PBS e Pipette and pipette tips e Known semen sample e Timer Each new lot of SERATEC PSA Semiquant Test devices must be subjected to the appropriate in house quality control t
28. 1030 FAD BIO SOP 3 1 Page7 of 8 Issued by Technical Leader Revision Date May 30 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 3 Equipment Quality Control and Maintenance 200 1000 3 250 242 5 257 5 3 750 727 5 772 5 3 1000 970 1030 FAD BIO SOP 3 1 Page 8 of 8 Issued by Technical Leader Revision Date May 30 2014 Effective Date June 1 2014 Forensic Biology Screening Ouality Assurance Forensic Analysis Division Standard Operating Procedures Biology 4 Guality Assurance 4 Guality Assurance In order to provide and maintain the guality of the work provided in the Biology section it is necessary to identify certain reagents as critical Critical reagents are those that reguire testing prior to use on evidentiary samples in order to prevent unnecessary loss of sample All reagent preparations and guality control testing must be documented on the appropriate worksheet or log If any critical reagent does not pass all quality control checks it may not be utilized in casework All inconsistencies will be documented and reported to the Section Supervisor Problems that cannot be resolved must be reported to the manufacturer Reagents and supplies that have passed their expiration dates may not be used on casework samples Outdated reagents may be used for training purposes only but must be clearly marked as such 4 1 Chemical and Reagent Labels Purchased chemicals and reagents will be marked on
29. 2014 Standard Operating Procedures Biology 4 Guality Assurance e SERATEC PSA Semiguant test devices 4 2 1 Acid Phosphatase AP Spot Test Scope AP Spot Test solution undergoes a color change Brentamine reaction in the presence of acid phosphatase which is found in highest concentration in semen Instructions for use and interpretation are in the AP Spot Test procedure Safety This reagent presents the following hazards may cause cancer irritant to eyes respiratory system and skin and corrosive can cause burns Wear gloves lab coat and mask during preparation and use Broken skin should be covered Equipment and Supplies e AP Spot Test premixed powder purchased e Darkened or foil wrapped container for storage e Scale e Sterile diH O e Timer e Known semen sample e Known non semen sample Preparation 1 Measure 10mL of sterile diH2O in an appropriate container 2 Add 0 26g of the purchased AP Spot Test premixed powder 3 Mix until AP Spot Test premixed powder is completely dissolved in water Note Volume may be smaller or larger than 10mL but the amount of AP Spot Test premixed powder added should be adjusted accordingly Prior to use each day the prepared AP solution must be subjected to the appropriate in house quality control test as outlined below 1 A known semen sample will be used as a positive control This in house prepared sample is typically presented on filter paper 2 An untreated piece of fi
30. 4 Standard Operating Procedures Biology 13 Chelex Extraction of DNA a Ensure the substrate is completely saturated Additional Chelex solution may be needed to completely submerge the swab b If additional Chelex is needed the same volume must be added to reagent blank Add 10 uL of Proteinase K and 15 6 uL of 1M DTT Vortex sample for 10 seconds Incubate at 56 0 C for 30 45 minute Vortex Incubate at 100 C for 8 minutes Take care to ensure that microcentrifuge tube lids remain closed during the 100 C incubation Briefly vortex then centrifuge the tube to pull beads to the bottom of the tube a Chelex beads and substrates should be pulled to the bottom of the tube Ifa supernatant layer is not visible then centrifuge again A higher centrifugation setting may be necessary A pipette tip may be used to force the substrate down prior to centrifugation b Alternatively if substrate will not stay in the bottom of the tube use a large bore pipette to remove at least 75 ul of supernatant to a new tube Centrifuge the new tube to pellet any beads Continue with step 9 9 Carefully transfer 50 uL of supernatant into a new 1 5ml microcentrifuge tube Be sure to not transfer any Chelex beads a If any of the beads are transferred pipette the supernatant back into the Chelex solution Repeat steps 7 9 Use a new final tube if supernatant is added back to the Chelex solution 10 Store samples refrigerated up to 2 week
31. Date June 1 2014 Standard Operating Procedures Biology 3 Equipment Quality Control and Maintenance 3 Record both the MQ cm reading and the temperature reading displayed on the resistivity monitor in the Water Quality logbook 4 If reading is below 15MQ cm call for service A qualified service technician may repair the system Label the system as being out of service until the appropriate service is performed and the instrument is functioning properly Balances Please refer to the Laboratory Quality Manual for the balance calibration schedule FAD BIO SOP 3 1 Page 6 of 8 Issued by Technical Leader Revision Date May 30 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 3 Equipment Quality Control and Maintenance Addendum 3A Volumetric Check Acceptable Check uL Pipette Range Percent Volume uL Average needs to be between these values 0 5 10 5 2 5 2 375 2 625 3 7 5 7 275 7 725 3 10 9 7 13 1 10 5 2 5 2 375 2 375 3 7 5 7 275 7 725 3 10 9 7 10 3 5 50 3 12 5 12 125 12 875 3 37 5 36 375 38 625 3 50 48 5 51 5 2 20 3 5 4 85 5 15 3 15 14 55 15 45 3 20 19 4 20 6 10 100 3 25 24 25 25 75 3 75 72 75 77 25 3 100 97 103 20 100 3 25 24 25 25 75 3 75 72 75 77 25 3 100 97 103 20 200 3 50 48 5 51 5 3 150 145 5 154 5 3 200 194 0 206 40 200 3 50 48 5 52 3 150 145 5 154 5 3 200 194 0 206 50 200 3 50 48 5 51 5 3 150 145 5 154 5 3 200 194 206 100 1000 3 250 242 5 257 5 3 750 727 5 772 5 3 1000 970
32. amiliarity with the Laboratory Safety Manual Any incident or condition that occurs in or under the control of the laboratory and that threatens the immediate or future health of any individual must be immediately brought to the attention of the section supervisor and a laboratory safety officer Corrective action related to safety incidents will be defined by laboratory management FAD BIO SOP 2 1 Page 3 of 3 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Forensic Biology Screening Eguipment Ouality Control and Maintenance Forensic Analysis Division Standard Operating Procedures Biology 3 Equipment Quality Control and Maintenance 3 Equipment Quality Control and Maintenance 3 1 Maintenance and Calibration In order to provide and maintain the quality of the work provided in the Biology section it is necessary to ensure laboratory equipment is in good working order Routine quality control and maintenance accomplishes this The calibration intervals listed below are generally considered to be the minimum appropriate in each case provided that the equipment is of good quality and of proven stability and the laboratory has both the equipment capability and expertise to perform adequate internal checks More frequent checks are acceptable If there is any question concerning the reliability of an instrument or piece of equipment a maintenance check should be performed immediately Full maintenance
33. as been deposited However on some substrates semen stains are not readily visible Under intense blue light 450 nm semen stains typically fluoresce Blood absorbs light and will not fluoresce fibers and other bodily fluids can typically fluoresce Should fluorescence take place the result only suggests the presence of semen the test is therefore a presumptive test Equipment Materials and Reagents e Alternate light source ALS set at 450 455 nm e Orange and or yellow glasses Procedure 1 Turn on power switch and ensure the fans are working Wear the proper glasses given and darken the examination room Turn on lamp switch Direct the ALS at the evidence samples Oi SL RO Document areas of apparent fluorescence on the evidence When ready to shut down the machine turn off lamp switch and wait for the unit to cool down 75 minutes Then turn off the power switch Interpretation FAD BIO SOP 7 1 Page 2 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 7 Semen Detection Should a stain fluoresce the result only indicates the possible presence of semen Substrate controls may be included in body fluid identification tests as appropriate 7 2 Acid Phosphatase Spot Test Scope Acid phosphatase AP is found in relatively large quantities in semen and its presence is indicative of the possible presence of semen in a stain In the following
34. ce Preparation Reagent B 1 Measure 250 mL of diH20 2 Add 6 5 g of sodium perborate 3 Mix until all of the powder is dissolved Mix equal volumes of Reagents A and B to create a luminol mixture The mixture is unstable and must be prepared fresh immediately before use it is only stable for about one hour Prior to use the luminol mixture must be subjected to the appropriate in house quality control test as outlined below Darken the examination room as completely as possible Spray luminol mixture onto a known blood sample prepared in house Spray luminol mixture onto a known non blood sample cotton swab or filter paper Observe samples for immediate luminescence Oe Se NS VE The known blood sample must give an immediate luminescence indicating a positive result 6 The cotton swab or filter paper must not produce luminescence indicating a negative result The results of each control test must be recorded on every applicable serology worksheet Storage Labeling and Expiration The luminol powder sodium carbonate and sodium perborate should be stored in a cool dry well ventilated area away from incompatible substances The expirations for reagents A and B stored separately are 8 weeks from date solutions were prepared If stored the labels must include at a minimum Luminol Reagent A or Luminol Reagent B as appropriate initials the date prepared and the expiration date Prior to each use check the reagen
35. ce of the High Dose Hook Effect may give false negative results due to the presence of a high concentration of human hemoglobin in the sample When huge amounts of human Hb bind to the antibody to form an antigen antibody complex but also free Hb migrates towards the test area T the antibody in the test area T is blocked by this free Hb Asa result the mobile antigen antibody complex with the pink color cannot bind to the antibody and the pink line cannot form in the test area T even though a lot of Hb is actually present In such cases where human blood is strongly suspected the extract may be retested using a 1 10 or 1 100 fold dilution 6 4 References 1 Abacus Diagnostics Inc ABAcard HemaTrace For the Forensic Identification of Human Blood Technical Information Sheet 2 Gaensslen Robert E 1983 Sourcebook in Forensic Serology Immunology and Biochemistry U S Department of Justice National Institute of Justice Sections 10 3 2 and 10 3 3 3 Lee Henry C 1982 Identification and grouping of bloodstains In Forensic Science Handbook Volume 1 Richard Saferstein ed Prentice Hall Inc Englewood Cliffs New Jersey Chapter 7 p 273 4 Lytle L T 1978 Chemiluminescence in the visualization of forensic bloodstains Journal of Forensic Sciences 23 3 550 562 5 Saferstein R Forensic Science Handbook Identification and Grouping of Bloodstains Prentice Hall Inc 1982 p 274 6 Stoilo
36. chnical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Guality Assurance 4 4 Proficiency Testing Proficiency testing and review will follow the requirements of the quality manual In addition the Quality Manager will maintain a copy of the analysis documentation for each proficiency test Proficiency tests will be analyzed and interpreted according to standard operating procedures including technical review Proficiency test participants will be notified of their final test results Analysts will enter into a proficiency test program within 6 months of being deemed competent on any portion of casework analysis Proficiency testing should include each technology to the full extent to which analysts and technicians participate in casework Proficiency work is to follow as closely as possible that of normal casework If symbols are used in the reporting of data to the proficiency testing agency they must be defined in the results submission form It is also advisable to include comments in the comments section of the proficiency results form to explain certain results For example if a result is reported as inconclusive the reason should be presented in the comments section During the case file reviews the proficiency results form including the screening data and comments sections along with the case file shall be reviewed by both the technical and administrative revie
37. ctly aligned in the holder Push the tray door to open it Insert the plate into the plate holder Load standard 96 well plates with the notched A12 position at the top right of the tray FAD BIO SOP 14 1 Page 4 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification Well A1 Close the tray door Starting the 7500 Real Time PCR System Overview Starting the 7500 Real Time PCR System involves 1 Starting the Computer if not already on 2 Powering on the Instrument 3 Starting the 7500 SDS Software Starting the Computer 1 Open the laptop computer by pushing in the front center button holding it and lifting the lid 2 Press the power button on the computer 3 Enterthe username associated with the computer if applicable 4 If required type the corresponding password in the password field NOTE Wait for the computer to finish starting up before powering on the 7500 instrument FAD BIO SOP 14 1 Page 5 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification Powering on the Instrument 1 Press the power button on the lower right front of the 7500 instrument 2 The indicator lights on the lower left of the front panel cycle through a power on seguence 3 When the green power indicator is lit not f
38. e 3 Mix until all of the powder is dissolved Procedure 1 Mix equal volumes of Reagents A and B to create a luminol mixture The mixture is unstable and must be prepared fresh immediately before use it is only stable for about one hour Darken the examination room as completely as possible 3 Spray luminol mixture onto evidence and observe for luminescence spraying will deposit a light white film on surfaces Document or photograph luminescent areas 5 Re spray as necessary The ability to further test the stain decreases with increased or repeated spraying Excessive spraying will cause stains to run therefore excessive spraying of non porous surfaces is not suggested 6 Dispose of excess solution in the regular sink flushing with water Interpretation Faint to strong luminescence shows oxidation of the luminol reagent and represents a positive presumptive result for blood A negative result a lack of luminescence indicates that blood is absent or below the detection threshold Substrate controls may be included in body fluid identification tests as appropriate 6 3 ABAcard HemaTrace Test Devices Scope The ABAcard HemaTrace test is a qualitative detection method specifically designed for forensic identification of human blood Heme is a part of the hemoglobin molecule that is characteristic of red blood cells found in blood The ABAcard HemaTrace has been shown to detect as little as 0 05 ug ml of hemoglobin
39. e 3 Press the push button to the first positive stop 4 Holding the pipette vertically immerse the tip into the sample liquid per manufacturer s recommended depth 5 Release the push button slowly and smoothly to aspirate the sample 6 Pausing for a moment withdraw the tip from the liquid 7 Depending on the sample it may be necessary to pre rinse the tip before aspirating the sample 8 To dispense the sample place the tip into the transfer vessel and press the push button to the first stop 9 Pausing fora moment press the push button to the second stop to blowout or expel any remaining liquid 10 Slowly release the push button 11 Eject the tip by pressing the ejector button Performance Verification Maintenance FAD BIO SOP 3 1 Page 4 of 8 Issued by Technical Leader Revision Date May 30 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 3 Equipment Quality Control and Maintenance 1 Each pipette should be externally calibrated and certified by an approved calibration vendor annually 2 Internal pipette verifications should be performed about 6 months after the external calibration 3 Pipette performance verification should be documented 4 To check for accuracy room temperature deionized water should be pipetted into a weighing vessel on an analytical balance 5 The pipette should be checked using 3 points over the full range with 5 replicates at each volume 6 Each pipette
40. e the reason s for the requested analyses and the quality and quantity of the evidence In order to expedite casework with the exception of sexual assault kits it is recommended that 5 10 items of evidence should initially be screened for cases containing large volumes of evidence Emphasis should be placed on those items believed to be of the most significant evidentiary value after consultation with the submitting party Of the items screened it is recommended that a maximum of 5 items of evidence continue on to DNA analysis initially Additional items may be analyzed at a later time Individual case needs may dictate that the initial quantity of examined items is greater than the recommended range presented above When submitted in conjunction with a sexual assault kit clothing evidence does not typically have to be examined if the kit components yield sufficiently positive samples Sufficiently positive is fluid and relevant to the case information Clothing should be examined if the corresponding sexual assault evidence kit does not reveal any positive probative evidence if there are multiple suspects or there is a recent consensual sexual partner Good judgment using the case information and or the client s specific requests should always supersede this guideline 5 2 Evidence Handling It is not possible to anticipate every situation that may arise or to prescribe a specific course of action for every case therefore the exam
41. e Buffered Saline 1X Scope PBS is used as an extraction agent for serological stains Instructions for use and interpretation are in the Phosphate Buffered Saline procedure FAD BIO SOP 4 2 Page 8 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Guality Assurance Safety This reagent presents the following hazards causes severe eye respiratory tract and skin irritation Avoid contact with skin and clothing Wear gloves lab coat and other proper laboratory attire Broken skin should be covered Eguipment and Supplies e OmniPur 10X PBS Premixed Powder e diH O e Graduated cylinder e Two sterile containers for storage Preparation 10X 1 Measure 1 liter of diH O 2 Add 100g of 10X PBS Premixed Powder 3 Mix until all of the powder is dissolved 4 Autoclave solution Preparation 1X 1 Measure 100mL of 10X PBS solution 2 Measure 900 mL of sterile diH2O 3 Mix 10X PBS and sterile diH2O Each new lot of 1X PBS must be subjected to an appropriate in house quality control test with the lot of SERATEC PSA Semiquant test currently in use See SERATEC PSA Semiquant test quality control procedure for further detail Storage Labeling and Expiration 1X and 10X PBS solutions can be stored at room temperature Label each bottle with appropriate concentration lot number and expiration date Expiration for both PBS concentrat
42. e standard curve b Results of melt curve investigations 17 Place a copy of the report containing the results in the case file Reporting and Sample Retention 1 Samples that have less than 5 x 10 0 005 ng ul of detectable male DNA present shall be reported as inconclusive 2 Inconclusive samples may proceed to DNA analysis depending on the availability of additional male DNA positive samples within a particular case FAD BIO SOP 14 1 Page 12 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014
43. e used to sterilize biohazard trash prior to discarding Please consult the instructions on the outside of the unit for specific information on operation of the autoclave Whenever an item is autoclaved a small piece of autoclave tape must be adhered to the item to verify correct functioning of the autoclave Use proper sterilizer loading procedures when placing materials in sterilizer chamber Those include all solid containers instruments must be placed so that water or air will not be trapped in them and sufficient space must be left between items to allow steam circulation The temperature gauge should read 120 C at the start and during the appropriate cycle If the autoclave is not functioning properly a qualified service technician may repair the instrument Alternate Light Source ALS Machines ALS machines are used to presumptively identify semen in forensic casework The fan air filter optical filters and lenses should be cleaned on a regular basis approximately every 6 months where applicable Please consult the appropriate equipment manuals for more specific instructions on maintenance and operation of the ALS machines Swab Drying Box E025 FAD BIO SOP 3 1 Page 3 of 8 Issued by Technical Leader Revision Date May 30 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 3 Equipment Quality Control and Maintenance Swab drying boxes are used to dry swabs created in the lab during forensic casework Before
44. ed In addition to the storage requirements detailed in the Quality Manual the following procedures will be followed Sexual assault kits may be stored in a cooler freezer or dry area at room temperature once received in the laboratory After analyses sexual assault kits may be stored at room temperature Sexual assault kits that contain blood or urine specimens are ideally stored in a refrigerator When applicable wet evidence should be dried upon receipt Refrigerate or freeze liguid whole blood specimens until a sample is dried on an appropriate substrate be careful when frozen blood has thawed as it may expand and cause vial breakage Portions of blood standards submitted in liguid form should be dried on stain cards within 30 days of receipt by the assigned analyst Once the sample is in dried form the liguid blood tube will be placed back in its original packaging and returned to the property room Cases containing small dry items may be stored at room temperature or frozen depending on available space Larger items such as clothing bedding weapons and other physical evidence containing bloodstains should be stored in a dry area at room temperature until examination FAD BIO SOP 5 1 Page 3 of 4 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 5 Evidence Evaluation and Handling Consumption of Evidence The evidence s quality and quantity will be pre
45. est as outlined below 1 Make a 1 10 dilution from a known semen sample in 1X PBS 2 Add 120 ul of dilution to sample well of one test device to be used as a positive control 3 Add 120 ul of 1X PBS to sample well of second test device to be used as a negative control Read results up to 10 minutes from application of sample 5 The 1 10 dilution of known semen must give a positive result The 1X PBS must give a negative result 6 Mark the stock solution bottle of 1X PBS with all lot numbers of SERATEC PSA Semiquant test devices quality control checked with that lot of 1X PBS Storage Labeling and Expiration The test devices must be stored at room temperature and must remain in the sealed pouch until use Label each test device with the name of the appropriate sample used The test devices may be used on casework samples up to the expiration date stated on the sealed pouch 4 3 Logbook Reagent preparation logs should include the lot number the reagent name the initials of preparer and the expiration date Quality control logs for ABAcard HematTrace kits Phosphate Buffered Saline 1X SERI Christmas Tree Stain and SERATEC PSA Semiquant tests will also contain the test date signature of the analyst performing the quality control a second reader signature if applicable and date and any supporting documentation necessary to demonstrate the reagent met all of the standards FAD BIO SOP 4 2 Page 11 of 12 Issued by Te
46. etection e Flood the slide with 95 ethanol and allow the slide to dry for at least 5 minutes on the heat block 3 Examine the slide for the presence of spermatozoa at 1000X magnification 4 Document the approximate number of sperm seen on the slide Once the count exceeds 25 spermatozoa observed the documented count can be 254 gt 25 or TNTC The presence of only one spermatozoon must be verified by a second qualified analyst who must initial and date the examination documentation 5 If too much cellular material and or debris is potentially obscuring any spermatozoa on the slide for the analyst to make a determination about the presence or absence of sperm the following procedure may be performed a Centrifuge the sample to pellet any cells Remove and save supernatant for p30 analysis b Wash the cell pellet 1x with 500ul of sterile diH2O or 1X PBS pipetting the pellet up and down or vortexing c Centrifuge again to pellet any cells d Re suspend in 50 to 300ul of sterile diH2O or 1X PBS again by pipetting up and down or vortexing a Use the new suspension to make another slide repeating steps 2 5 Examination documentation must reflect the creation of multiple slides INTERPRETATION Sperm heads stain red and the tails stain green by this procedure A minimum of one sperm head should be identified to confirm the presence of semen A search of the entire slide is required before negative results
47. for spermatozoa can be reported If the slide contains too much cellular material or debris for the analyst to determine the presence or absence of sperm the analyst may then report the result as inconclusive Alternatively if sperm like objects are observed but one or more of the identifying characteristics are lacking the slide may be reported as inconclusive It is recommended that inconclusive samples proceed to DNA analysis along with positive samples 7 5 SERATEC PSA p30 Semiquant Test Scope The cells that line the ducts of the prostate make a glycoprotein known as p30 or prostate specific antigen PSA The protein is secreted into seminal fluid to a concentration of approximately 0 2 3 0 mg ml and its detection may indicate the presence of semen PSA may be found at very low concentrations in vaginal fluid 0 4 1 25 ng ml and is therefore a presumptive test FAD BIO SOP 7 1 Page 7 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 7 Semen Detection The SERATEC PSA Semiguant test is a detection method specifically designed for forensic identification of p30 This test will be used gualitatively only in this laboratory The test is capable of detecting p30 in a concentration range of at least 2ng ml 100ug ml PSA SERATEC PSA Semiquant test contains two monoclonal murine anti PSA antibodies as active compounds One of
48. g Clean work surfaces in the evidence examination areas thoroughly with a lab approved decontaminant prior to and at the end of each evidence examination session Use disposable bench paper whenever possible and change each time it comes in contact with evidence Use a clean cutting surface such as weighing paper or piece of paper for evidence Protect supplies of this paper from dust and other particulates or aerosols Clean instruments e g scalpels scissors forceps with decontaminant between evidence samples To prevent contamination of other standards or evidence handle each piece of evidence one at a time Liquid samples such as blood standards and wet exhibits shall be allowed to air dry in a method that it will not come into contact with other pieces of evidence Wear gloves when cleaning glassware and plastics In general clean glassware plastics with an appropriate soap e g Liquinox or Alconox and water Rinse with deionized or distilled water and allow to air dry inverted Glassware should be autoclaved Plastic ware should be placed under UV light for 15 minutes Store glassware and plastic ware after being autoclaved or being cleaned by UV light FAD BIO SOP 2 1 Page 2 of3 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 2 Facilities 2 3 Safety There are biological and chemical hazards in the laboratory Each lab employee is responsible for f
49. he intensity of this line correlates with a p30 concentration of approximately 4 ng ml It is expected for all tests and must be present The absence of the internal standard line is also an invalid result and the test should be repeated if adequate sample remains 3 Test T line A line indicates the presence of semen and should be documented as a positive result The absence of a line or a line that appears after 10 minutes is a negative result or p30 concentration is below the detection limit Samples that produce a negative result but show strong positive acid phosphatase activity must be diluted to an appropriate dilution and be re tested to ensure the High Dose Hook Effect is not occurring see SOP 6 3 ABAcard HemaTrace Test Devices 7 6 Guidelines for Sexual Assault Cases When Contact or Digital Penetration is Alleged It is not possible to anticipate every situation that may arise or to prescribe a specific course of action for every case therefore the examiner must exercise good judgment based on experience and common sense The following are intended only as guidelines for cases in which digital penetration or other contact may have occurred Specific case information may suggest a course of action other than what is prescribed below Competent ex can clearly recall incident External swabs with clear notes on why it was collected ex he kissed my breast e Bathed NTC add bathed statement in report e Not bathed
50. indicated by the formation of an additional line Thus a PSA positive sample will show three colored lines in the result window Equipment Materials and Reagents 1 SERATEC PSA Semiquant test device one per sample 2 Body fluid extract 3 Pipettes and pipette tips 4 Timer Procedure 1 Allow previously extracted sample s to warm to room temperature if they have been refrigerated For each sample unwrap and label a SERATEC PSA Semiquant test device Add 120 ul of the supernatant to the sample well of the device Results may be read up to 10 minutes from application of the supernatant Record the SERATEC PSA Semiquant test results on the Serology Results worksheet Have a second qualified analyst examine the SERATEC PSA Semiquant test device and verify the documented test result s by initialing and dating the appropriate Serology Results worksheet DARON Interpretation There are three lines that appear in the test device result window FAD BIO SOP 7 1 Page 8 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 7 Semen Detection 1 Control C line the presence of this line confirms the integrity of the test components and the test procedure It is expected for all tests and must be present Absence of the C line is an invalid result and the test should be repeated if adequate sample remains 2 Internal Standard line T
51. iner must exercise good judgment based on experience and common sense In some cases the manual offers guidelines for analysis that must be tempered with the experience of the examiner However any portion of a procedure not explicitly qualified as a guideline may not be modified for use in casework without prior written approval as outlined in the Quality Manual On a sexual assault case for example the analyst will have a choice of using conventional serology for the detection of semen or quantitative PCR for the detection of male DNA The evidence type may warrant one method over another It may be more informative to detect actual spermatozoa on a non intimate garment that may have been collected from a common area than to detect male DNA alternatively the detection of male DNA via quantitative PCR on an intimate swab may be more probative to a particular case When ample sample permits the analyst should consider retesting when inconclusive results are obtained Please also refer to the Quality Manual for the Handling of Evidence 5 8 FAD BIO SOP 5 1 Page 2 of 4 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 5 Evidence Evaluation and Handling Universal Precautions Body fluids and extracts may contain infective agents All analysts should routinely use appropriate barrier precautions to prevent skin and mucous membrane exposure when in contact wi
52. ing present when this test is used as forensically the practical implications of this cross reactivity is minimal The ABAcard HemaTrace test may be skipped if in the analyst s opinion consumption for human origin testing will reduce the potential success of subseguent DNA analysis Samples that are of limited size may be documented as Quantity Not Sufficient For Further Analysis QNS Only a presumptive screening result will be reported but DNA analysis may be performed Eguipment Materials and Reagents e ABAcard HematTrace Test device one per sample e ABAcard HematTrace extraction buffer e Pipettes and pipette tips e Timer Procedure 1 Allow the sample s to warm to room temperature if they have been refrigerated 2 Extract body fluid by placing suspected bloodstain sample into the extraction buffer provided with the kit and allow to sit for 5 30 minutes Aged samples may be less soluble and should therefore be soaked closer to 30 minutes The extract s may be placed at room temperature in an ultrasonic cleaner for 5 15 minutes The analyst must exercise good judgment regarding the amount of sample to be portioned for each ABAcard Hematrace test device providing sufficient sample for subsequent DNA analysis Stain intensity and size are a few characteristics that should be considered Generally up to approximately 1 4 of a swab or up to approximately 1 cm of a stain may be consumed for this analys
53. ing the DNA Quantification Standards Make a standard curve dilution series consisting of Standard ng uL Male Genomic DNA Standard 50 B 10 C 2 D 0 4 E 0 08 F 0 016 G 0 0032 To create this series of standards perform the following tasks FAD BIO SOP 14 1 Page 2 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification Label 6 microcentrifuge tubes B C D E F and G 2 Dispense 40 uL of TE into the six microcentrifuge tubes 3 Vortex the Male Genomic DNA Standard for 3 to 5 seconds a Allow the Male standard to warm to room temperature b If this is the first use of the standard thaw the DNA 4 Prepare Standards B through G a Pipette 10 uL of the Male Genomic DNA Standard to the tube for Standard B b Mix the dilution thoroughly c Using a new pipette tip add 10 uL of the prepared standard to the tube for the next standard e g 10 uL of Standard B into the tube for Standard C the resulting mixture will contain 10 uL of the previous standard and 40 uL of TE d Mix the dilution thoroughly e Repeat steps 4c and 4d until the dilution series is complete NOTE Any unused standard can be labeled with the analyst s initials and date of creation e g ABCO10101 and stored at 2 C to 8 C for up to two weeks Reaction and Plate Setup Procedure 1 Allow the Plexor HY kit componen
54. ions is one year from the date 10X PBS solution was prepared Both concentrations of PBS may be used on casework samples up to this expiration date 4 2 7 SERI Christmas Tree Stain Scope SERI Christmas Tree Stain is a Kernechtrot Picroindigocarmine differential biological stain that assists in scanning slides for the presence of spermatozoa The test consists of a two stain method which dyes sperm heads red tails green and epithelium green or blue with red nuclei Instructions for use and interpretation are in the SERI Christmas Tree Stain procedure FAD BIO SOP 4 2 Page 9 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Quality Assurance Safety This reagent presents the following hazards May cause irritation upon contact Toxic Do not swallow Do not inhale Avoid contact with skin and eyes Wear gloves and proper laboratory attire Broken skin should be covered Eguipment and Supplies Each Microscope slide Known semen sample Pipette and pipette tips Heat block Christmas Tree Stain Solution A and Solution B Timer Tap water 95 ethanol Compound microscope new lot of SERI Christmas Tree Stain must be subjected to the appropriate in house guality control test as outlined below 1 Pipette 10 ul of a neat in house sample onto a glass slide Fix smear by heating the slide on the heat block until dry Stain
55. is 3 For each sample unwrap and label an ABAcard HematTrace test device 4 Add 150 ul of supernatant to the sample well S of the device 5 Results may be read up to 10 minutes from application of the supernatant Positive results can be seen as early as 2 minutes 6 Record the ABAcard HemaTrace test results on the Serology Results worksheet 7 Have a second qualified analyst examine the ABAcard HemaTrace test device and verify the documented test result s by initialing and dating the appropriate Serology Results worksheet Interpretation FAD BIO SOP 6 1 Page 5 of 6 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 6 Blood Detection There are two lines that appear on the test device 1 Control C line the presence of this line confirms the integrity of the test components and the test procedure It is expected for all tests and must be present Absence of the C line is an invalid result and the test should be repeated if adequate sample remains 2 Test T line A line indicates the presence of human hemoglobin and should be documented as a positive result The absence of a line or a line that appears after 10 minutes is a negative result A negative result indicates that blood is absent or below the detection threshold Substrate controls may be included in body fluid identification tests as appropriate Note the presen
56. lashing communication is established between the computer and the instrument NOTE If the green power on indicator is flashing or the red error indicator is lit see the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide PN 4347828 Power button Indicator lights Starting the 7500 SDS Software 1 Start the 7500 System Software Double click on the software icon 2 The software starts and displays the word Disconnected in the status bar on the bottom right corner The status changes to Connected only after the New Document Wizard is completed the software is initialized and the software is connected to the 7500 instrument If the connection is successful the software displays Connected in the status bar on the bottom right corner Creating a Plate Document and Starting the Run 1 Click File gt New 2 Click Browse below the Template box FAD BIO SOP 14 1 Page 6 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and Assay Absolute Guantification Standard Curve Container 96 Well Clear Template Blank Document Browse Operator Ihalsell Comments SDS v1 2 RN Plate Finish Cancel 3 Na
57. le cotton swab s e Known blood sample e Known non blood sample Prior to use each day PHT Solutions B and C must be subjected to the appropriate in house quality control test as outlined below 1 Rub a cotton swab moistened with sterile diH2O on a known bloodstain sample This swab will be used as a positive control 2 An untreated cotton swab moistened with sterile diH2O will be used as a negative control Apply 1 drop of Solution B to each swab Apply 1 drop of Solution C to each swab Observe the swabs for a color change oP es wp The cotton swab with the known bloodstain sample must give an immediate pink color change indicating a positive result 7 The untreated cotton swab must have no color change indicating a negative result The results of each control test must be recorded on every applicable serology worksheet Storage Labeling and Expiration PHT test kits can be stored at room temperature Each analyst will have an aliquot of Solutions B and C that will be placed in amber bottles stored at room temperature or refrigerated Each bottle will be labeled with appropriate reagent name lot numbers and expiration dates The kits may be used on casework samples up to the expiration date stated on the bottles FAD BIO SOP 4 2 Page 7 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Guality Assurance 4 2 5 ABAcard HemaTrace
58. lter paper or swab moistened with sterile diH2O will be used as a negative control Ideally the negative control substrate mimics your sample substrate as closely as possible 3 Apply 1 drop of prepared AP solution to both controls Observe samples for 60 seconds for a color change 5 The known semen sample must give a strong purple color change indicating a positive result FAD BIO SOP 4 2 Page 3 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Guality Assurance 6 The untreated filter paper or sterile swab must have no color change indicating a negative result The results of each control test must be recorded on every applicable serology worksheet Storage Labeling and Expiration Store prepared solution in a darkened or foil wrapped container Label with reagent name lot number and expiration date Reconstituted reagent is stable and sensitive for one day s use make solution fresh daily Store purchased powder frozen Purchased powder may be used on casework samples up to the expiration date stated on the bottle 4 2 2 Alternate Light Source Scope An Alternate Light Source is specially designed for detection of forensic stains fibers and fingerprints Instructions for use and interpretation are in the Alternate Light Source procedure Safety This instrument presents the following hazards exposing the skin to the beam
59. lts of each control test must be recorded on every applicable serology worksheet Storage and Expiration Store eguipment in a clean dry atmosphere The bulb should be replaced after 3 3 5 years of non regular usage 4 2 3 Luminol Scope Catalytic tests for blood are based on the peroxidase like activity exhibited by the heme group of hemoglobin In the case of luminol the catalytic oxidation of the substrate compound produces light Instruction for use and interpretation are in the Luminol procedure Safety This reagent presents the following hazards luminol is an irritant Sodium perborate and sodium carbonate are toxic and irritating Avoid breathing dust do not get in eyes on skin or on clothing Avoid breathing sprayed solution Wash hands after handling Gloves must be worn during testing Broken skin should be covered Equipment and Supplies e Luminol powder 3 aminophthalhydrazide e Sodium carbonate e Sodium perborate e Sterile diH O e Two darkened or foil wrapped spray bottles e Scale e Known blood sample e Known non blood sample Preparation Reagent A 1 Measure 250 mL of diH O 2 Add 2 5 g of sodium carbonate 3 Mix until all of the powder is dissolved 4 Add 0 5 g of luminol powder 5 Mix until all of the powder is dissolved FAD BIO SOP 4 2 Page 5 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Guality Assuran
60. ndard Operating Procedures Biology 10 Portioning for DNA Analysis Clothing 10 spermatozoa or p30 only 1 cm cutting 2 10 spermatozoa 0 5 cm cutting HemaTrace 0 5 1 cm cutting QNS V2 of the stain 3 Place portion into a microcentrifuge tube 4 Label microcentrifuge tube with case number item number and initials FAD BIO SOP 10 1 Page 3 of 3 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Forensic Biology Screening Case Records Forensic Analysis Division Standard Operating Procedures Biology 11 Case Records 11 Case Records 111 Examination Documentation The notes and other documentation must support the conclusions of the examiner The laboratory report must communicate both the analytical results and the conclusions of the examiner conveying the essence of what he or she would say if asked for an expert opinion in court Decisions may be made by police officers attorneys and the courts based on the report alone without examiner clarification so the report should be able to stand alone Some case record requirements can be found in the Quality Manual any additional case record reguirements are included here Refer to the guality manual for chain of custody policies and procedures and documentation of chain of custody as well as documentation reguired in all Laboratory case records Documentation must be in such a form that another gualified examine
61. negative samples 6 1 Phenolphthalein Test Kit Scope Catalytic tests for blood are based on the peroxidase like activity exhibited by the heme group of hemoglobin The test is exceedingly sensitive to minute traces of hemoglobin and its derivatives but will produce a false positive reaction in the presence of any of a number of oxidizing substances Should a color reaction take place the result only indicates the possible presence of blood the test is therefore a presumptive test Safety This reagent presents the following hazards Solution B Flammable Handle with care Harmful or fatal if swallowed inhaled or absorbed through skin Avoid contact with eyes skin and clothing Light sensitive Avoid light heat sparks and flame Keep container tightly closed Solution C Handle with care May be harmful if swallowed inhaled or absorbed through skin Avoid contact with eyes skin and clothing Avoid heat sparks and flame Keep container tightly closed Wear gloves lab coat and mask during preparation and use Broken skin should be covered Equipment Materials and Reagents e Sterile diH2O e Sterile cotton swab s or filter paper e Phenolphthalein Forensic Test reagents Solutions B and C Solution A is also included in the Phenolphthalein Forensic Test However previous testing showed that using sterile diH2O in place of Solution A did not affect the outcome of this test Therefore sterile diH2O will be used in place
62. ntire kit at 15 C to 25 C upon receipt Store the male genomic DNA standard at 2 C to 8 C after first thaw the remaining kit should continue to be stored at 15 C to 25 C between uses Plexor HY System 200 reactions Product Size Cat DC1001 Includes 2x imi Plexor HY 2X Master Mix e 200ul Plexor HY 20X Primer IPC Mix e 150ul Plexor HY Male Genomic DNA Standard 50ng ul e 2 x 1 25ml Water Amplification Grade Plexor HY System 800 reactions Product Size Cat DC1000 Includes 8 x 1mi Plexor HY 2X Master Mix e 4x 200ul Plexor HY 20X Primer IPC Mix 3x 150ul Plexor HY Male Genomic DNA Standard 50ng ul e5 x 1 25ml Water Amplification Grade FAD BIO SOP 14 1 Page 1 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification NOTE Keep Primer IPC Mix and Master Mix protected from direct exposure to light Excessive exposure to light may affect the fluorescent dyes Creating a Plexor HY worksheet 1 Open the Plexor HY Worksheet template an Excel spreadsheet 2 Type sample names into the sample sheet Ensure each standard is present and in duplicate 3 Print the file for documentation in the case file s 4 Save the excel file with initials and date of run i e AB092012 5 Close excel Prepar
63. ocedures Biology 14 Plexor HY Guantification iii Check Melts displayed for sample designated as unknowns NOTE Check messages will only be displayed on samples that have concentration values The message indicates that the melt curve needs to be assessed for that sample b Examine the melt curves for samples that are NOT Ok i Melt curves need to be assessed for both Autosomal and Y results c Melt curves can be examined under the PCR Curves tab The melt curve is the bottom graph NOTE Melt curves are a measure of the PCR products produced during amplification If a sample has a low level DNA concentration but no melt curve then the detected fluorescence is most likely from non specific fluorescence and not from amplifiable DNA d When examining the melt curves in the PCR Curves tab three different calls are available in the Tm column on the right side i Yes the melt curve is within the designated range ii No the melt curve is not within the designated range iii No Call the melt curve is within the designated range but there is insufficient amplification product to cross the threshold e Documentation of why melt curves were not OK will need to be made on the report print out 3 Review raw data for all samples and controls Examine amplification curves for unusual morphology Samples with unusual curves may be re run or reported as inconclusive NOTE It is recommended tha
64. on2 htm Accessed July 21 2012 Edwin L Jones Jr 2005 The Identification of Semen and Other Body Fluids In R Saferstein Ed Forensic Science Handbook Volume II pp 329 399 Mt Laurel New Jersey Pearson Prentice Hall Baechtel S F 1988 The identification and individualization of semen stains In Forensic Science Handbook Volume 2 Richard Saferstein ed Prentice hall Inc Englewood Cliffs New Jersey Chapter 7 p 349 Gaensslen Robert E 1983 Sourcebook in Forensic Serology Immunology and Biochemistry U S Department of Justice National Institute of Justice Sections 10 3 2 and 10 3 3 Kind Stuart S 1957 The use of acid phosphatase in searching for seminal stains Journal of Criminal Law Criminology and Police Science 47 5 597 600 Omniprint 1000A 110 Operating Instructions SERATEC PSA Semiguant Test In vitro diagnostic test for professional forensic use for the detection of seminal fluid by the semi guantitative determination of PSA Prostate specific antigen Product insert revised June 2011 Smith Charles 1979 An acid phosphatase reagent for use in searching large areas for seminal stains Unpublished 10 Stoilovic M 1991 Detection of semen and blood stains using Polilight as a light source Forensic Science International 51 289 296 11 Stone I C 1972 Staining of spermatozoa with Kernechtrot and picroindigocarmine for microscopical identification Document CIL No 2 S
65. outhwestern Inst Forensic Sci Criminal Investigation Laboratory USA FAD BIO SOP 7 1 Page 10 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Forensic Biology Screening Trace Observation Collection Forensic Analysis Division Standard Operating Procedures Biology 8 Trace Observation Collection 8 Trace Observation Collection 8 1 Scope This procedure may be used when necessary for the preservation of trace evidence prior to forensic biology analysis Examples of typical trace evidence in criminal cases include fingerprints glove prints hairs cosmetics plant fibers mineral fibers synthetic fibers glass paint chips soils botanical materials gunshot residue explosives residue and volatile hydrocarbons arson evidence However the reporting of trace may be limited to apparent hairs and fibers The Locard Exchange Principle presumes that when a person comes into contact with an object or person a cross transfer of material sometimes trace evidence occurs While this laboratory does not currently examine trace evidence it does make every effort to collect and preserve it for potential future analysis To do so trace evidence is generally collected prior to the commencement of biological analysis Dedicated trace collection room doors should remain closed at all times The rooms will be vacuumed and the floors damp mopped at least once per week when in use Lab coats will be
66. procedure acid phosphatase is detected by a color change reaction Acid phosphatase liberates the phosphate from a naphthyl phosphate and the released naphthol combines with tetrazotized o dianisidine to form a purple azo dye The AP test is semi quantitative A stronger reaction is more likely to indicate semen However because acid phosphatase occurs in other body fluids most notably vaginal secretions this is only a presumptive test The presence of semen in the sample can subsequently be confirmed by the presence of spermatozoa Equipment Materials and Reagents e Sterile diH2O e Sterile cotton swab s or filter paper e AP Spot Test reagent e Timer e Non porous surface e Clips optional AP Procedure for Swabs Note Begin at step 1 when it is necessary to transfer the suspected semen sample from an item of evidence to a cotton swab or swabs for ease of testing Otherwise begin at step 3 following the procedure below 1 Moisten cotton swab s with sterile diH2O Swab the suspected semen stain from the evidence Moisten filter paper with sterile diH2O Press the swabs firmly onto the filter paper Apply one or two drops of the AP Spot Test reagent to the filter paper Observe the treated filter paper for up to 60 seconds for a color change AO OE SR TSAR After interpretation document the test results onto the serology worksheet s 8 Discard used filter paper by placing it in the biohazard trash AP Mapping Procedure
67. r or supervisor in the absence of the primary examiner would be able to evaluate what was done and interpret the data The reviewer of the case should be able to determine from the notes that sufficient testing relevant testing and correct methods of testing were used To this end all documentation of procedures standards and controls used observations made results of tests performed charts graphs photographs sketches etc that are used to support the examiner s conclusions must be preserved as a record Observations data and calculations shall be recorded at the time they are made the date of an in house photograph shall be included on the photograph or associated examination documentation Examination records shall be of a permanent nature If a written examination record is created or if original observations are made on non traditional media for example sticky notes paper towels gloves etc then either the original media will be retained or an electronic scan picture of the original will be retained in the case record Once an electronic scan picture is created the original hardcopy may be destroyed Examination documentation should reflect the name and or initials of the individual who performed the work Appropriately completed SOP worksheets should be used during the analyses Examination documents should have notes that help in the identification of the item of evidence A written description may suffice for some items whe
68. ration Gloves must be worn during testing Clothing may protect unbroken skin broken skin should be covered Standards Control and Calibration One reagent blank must be processed for each extraction batch as a negative control Please refer to SOP 5 Evidence Evaluation and Handling for guidelines on general evidence handling and the consumption of evidence in particular Equipment Materials and Reagents e scissors scalpel tweezers e 5 Chelex e Proteinase K 10 mg ml e 1MDTT e tubes 1 5 ml microcentrifuge e microcentrifuge with rotor for 2 ml tubes at room temperature e water bath or dry bath 56 0 C e water bath or dry bath 100 0 C e vortex e stir plate Procedure Portioning A portion of all swabs provided will be taken for Chelex extraction Set the evidence onto paper Portion 1 8 of all swabs for a particular piece of evidence Place all portions into a microcentrifuge tube Label microcentrifuge tube with case number item number and initials PON Chelex Extraction 1 Make sure Chelex beads are evenly distributed in solution a If necessary re cap and mix stock by inversion periodically during processing b Alternatively chelex solution may be made in a sterile bottle and a reagent stir bar used to keep the beads distributed 2 Add approximately 200 uL of 5 Chelex solution FAD BIO SOP 13 1 Page 2 of 3 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 201
69. reas others may need a drawing sketch or photograph If an item is submitted for immediate analysis such as a mobile phone that will be swabbed for possible contact DNA and immediately returned to the submitting party it is not necessary for the evidence to be sealed and it may not be possible to establish a proper seal and or to be marked with a unique identifier However case record documentation must include identifying information that will permit a later identification of the item handled For example in the absence of analyst markings on the actual item of evidence a photograph of the mobile phone s serial number along with a photo of the mobile phone will enable later identification of the actual item handled Items collected at autopsy do not always include the name of the complainant as it may be unknown at the time of the autopsy If morgue evidence is received without the name of the complainant the evidence should be described using the Medico Legal number ML and or unknown Alternatively if written notice is provided by the investigator or other case agent the complainant name provided may be used in the evidence descriptions in conjunction with the FAD BIO SOP 11 2 Page 2 of 4 Issued by Technical Leader Revision Date July 19 2014 Effective Date July 19 2014 Standard Operating Procedures Biology 11 Case Records ML This written notice must be maintained in the case file and may be referred to in the
70. report Each examination worksheet s should include the following initials case number controls dates of analyses item names numbers quantity results and disposition Items that are not tested or opened should be described to the extent to which they can be A written description may suffice for some items whereas others may need a drawing or photograph Descriptions of evidence should be clear and sufficient to permit the later identification of an item They may include any of the following tears cuts missing buttons possible bullet holes location and length of possible stab holes soiling condition degree of degradation type of material significant foreign material etc Significant stains must be drawn or documented in a manner which clearly demonstrates the location relationship to other stains reactions to screening tests etc Abbreviations may be used see SOP 12 for a listing of approved abbreviations If needed the location of the substrate blank control and its relationship to the body fluid stain in question must be clearly documented The type of material or fabric the stain is on may be significant 11 2 Reports Report the results of any tests performed If presumptive tests are positive but confirmatory tests are not performed this fact should be reported For some items it may be useful to report what area of the item was tested Also include trace examination whether collected or observed Instructions should
71. s or frozen gt 2 weeks until ready to perform Plexor HY analysis Or Ole oo FAD BIO SOP 13 1 Page 3 of 3 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Forensic Biology Screening Plexor HY Ouantification Forensic Analysis Division Standard Operating Procedures Biology 14 Plexor HY Guantification 14 Plexor HY Guantification The Promega Plexor HY kit contains all necessary reagents for the simultaneous real time PCR amplification detection and guantification of human autosomal and human male DNA in a sample The results can be used to determine the presence of male DNA in samples with overwhelming female DNA The DNA guantification assay combines three separate assays e A multicopy human autosomal DNA assay e A multicopy human male DNA assay e An internal PCR control assay The kit works with the Applied Biosystems 7500 Real Time PCR System and SDS Software v1 2 3 for data collection Data analysis is accomplished by exporting the data to the Plexor Analysis Software v1 5 6 7 Safety Body fluids tissues and extracts may contain infective agents Use universal precautions during evidence handling Follow instructions for reagent preparation Gloves should be worn during testing Clothing may protect unbroken skin broken skin should be covered Kit Contents and Storage The Plexor HY kit comes in two sizes 200 and 800 reactions with a 20 uL reaction volume Store the e
72. s laboratory capacity volume of specific requests and sectional productivity FAD BIO SOP 11 2 Page 3 of 4 Issued by Technical Leader Revision Date July 19 2014 Effective Date July 19 2014 Standard Operating Procedures Biology 11 Case Records The following is a summary of how totals for this form should be calculated Category How to Calculate Number of Items Number of items listed on the report ALS Exams Number of items viewed with ALS excluding controls AP Number of swabs stains tested excluding controls Sperm searches Number of slides viewed p30 Number of cards tested PHT Number of swabs stains tested excluding controls HemaTrace Number of cards tested Trace collection observation Number of items with trace collected or trace observed Bloodstain cards Number of bloodstain cards made Swabbings cuttings Number of cuttings or swabs collected Portions for DNA Number of portions created Total examinations Total of examinations LIMS will calculate FAD BIO SOP 11 2 Page 4 of 4 Issued by Technical Leader Revision Date July 19 2014 Effective Date July 19 2014 Forensic Biology Screening Abbreviations Forensic Analysis Division Standard Operating Procedures Biology 12 Abbreviations 12 Abbreviations In addition to standard scientific abbreviations as well as those found in the dictionary the following abbreviations are defined here and acceptable for use
73. served as much as possible without sacrificing the guality of the analyses Good judgment must be exercised to determine the smallest amount of sample that should be consumed for analysis that will still provide accurate results Whenever possible at least half of the evidence sample will be preserved for possible re analysis for both additional screening and additional DNA analysis When this is not possible the appropriate personnel submitting officer prosecuting attorney and or defense attorney will be consulted and written permission will be obtained before the evidence is consumed All evidentiary hairs are treated individually and cannot proceed to DNA analysis without consumption permission Chain of Custody Refer to the Laboratory Quality Manual for chain of custody policies and procedures Reference Samples Whenever possible reference samples should be collected and retained from suspects complainants witnesses consensual sex partners and or other individuals for the purposes of elimination Oral swabs are the preferred reference sample for the Laboratory but blood is certainly a viable source of reference material Anecdotally Sexual Assault Nurse Examiner SANE protocol dictates that evidentiary oral swabs are not only collected well before reference known saliva swabs but also after oral rinsing to help prevent possible foreign DNA from contaminating a reference known saliva sample Potential for Contact DNA Should con
74. should fall within the verification tolerance ranges according to Addendum 3A before it may be used for casework 7 Ifa pipette fails a performance verification check it should be recalibrated Calibrations are not performed in house 8 Follow manufacturer s instructions for troubleshooting maintenance if needed 9 Ifan analyst has reason that a pipette is not working properly they must a Perform a pipette verification and if the pipette is not in proper working order b Clearly mark the pipette OUT OF SERVICE c Inform the section manager No laboratory case work will be performed using the pipette until the problem is corrected d Repair or send out the pipette for repairs e Verify that the pipette is working correctly and falls with in the proper tolerances following routine repair maintenance or calibration f Maintain the appropriate documentation and update the log book Water Filtration System Water is used in reagents that are prepared in the laboratory Therefore it is necessary to ensure only high quality reagent grade water is being utilized Generally 15MO cm or greater deionized water is sufficient for the reagents utilized in our laboratory Water quality should be checked on a weekly basis The following procedure will be utilized 1 Turn on the deionized water 2 Allow water to run for 1 2 minutes FAD BIO SOP 3 1 Page 5 of 8 Issued by Technical Leader Revision Date May 30 2014 Effective
75. t the nearest standards above and below the unknown sample be used for comparison of curve morphology 4 Return to the Forensic tab under the Reports tab 5 Check that all negative controls including reagent blanks have less than 5 x 10 0 005 ng ul of detectable DNA present If negative control values exceed 0 005 ng ul then consult with the Technical Leader on available options 6 Close the Plexor run file by File gt Close Current Analysis a Alternatively you can click the red X in the upper right run file window This is just above the printer icon 7 Save results if asked to do so 8 Reopen the file by File gt Open Analysis File FAD BIO SOP 14 1 Page 11 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification a Alternatively you can click the file icon in the menu bar 9 Navigate to the saved file and select it Click Open 10 Return to the Forensic tab underthe Reports tab if not already there 11 Make sure all samples are selected All samples will be highlighted blue 12 Select the print icon on the right 13 Under Page Setup change the orientation to Portrait 14 Click Print 15 Close the Plexor run file and software a Save any changes if prompted to do so 16 Add any comments to the report that are necessary Examples include a If any points were dropped from th
76. tact DNA potentially be probative in a case it is permissible for the examiner to bypass certain procedures that could possibly remove contact DNA that may be present For example an examiner may choose to skip the presumptive Acid Phosphatase press out test and simply portion for the confirmatory microscopy examination in an effort to preserve potential contact DNA if both penile and digital penetrations are alleged Examiners may also choose to skip serological analysis entirely in an effort to maximize the available DNA Case records must clearly document when and why a routine procedure such as the acid phosphatase or phenolphthalein press out test is not performed FAD BIO SOP 5 1 Page 4 of 4 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Forensic Biology Screening Blood Detection Forensic Analysis Division Standard Operating Procedures Biology 6 Blood Detection 6 Blood Detection Please refer to SOP 4 Reagent Quality Control for quality control testing storage labeling and expiration guidelines of the Phenolphthalein Test Kit Luminol and ABAcard HemaTrace Test Devices Many samples submitted to the Crime Laboratory are tested for the presence of blood Generally a sample will be subjected to a presumptive phenolphthalein test If positive the sample will be subjected to a confirmatory test for the presence of human blood Testing is typically discontinued on phenolphthalein
77. tection threshold Substrate controls may be included in body fluid identification tests as appropriate 7 3 Body Fluid Extraction Scope Possible body fluid stains are removed from the substrate and dissolved in PBS for testing The extract is used to collect cellular debris from a stain for spermatozoa examination and can also be used for p30 testing Microscopic examination for spermatozoa or p30 testing may be skipped if in the analyst s opinion consumption for these tests will reduce the potential success of subseguent DNA analysis Samples that are of limited size may be documented as Quantity Not Sufficient for Further Analysis QNS Only a presumptive screening result will be reported but DNA analysis may be performed Eguipment Materials and Reagents e Microcentrifuge tube s FAD BIO SOP 7 1 Page 4 of 10 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 7 Semen Detection e Forceps optional e Scalpel and or scissors e Mini centrifuge e 1X PBS e Pipette and pipette tips e Ultrasonic cleaner Procedure 1 Add sample to a microcentrifuge tube The analyst must exercise good judgment regarding the amount of sample to be portioned for stain extraction providing sufficient sample for DNA analysis Stain intensity and size are a few characteristics that should be considered Generally up to approximately Ya of a swab or up to approximatel
78. th any body fluids Gloves should be worn and changed after contact with each piece of evidence Masks lab coats and protective eyewear should be worn during procedures that anticipate contact with body fluids to prevent exposure of mucous membranes of the mouth nose and eyes All analysts should take precautions to prevent injuries caused by scalpels or other sharp instruments or devices during procedures when cleaning or during disposal After sharp instruments are used they should be placed in a puncture resistant container for disposal Evidence Reguiring Latent Print Examination Submitting parties may reguest that items be analyzed for latent prints as well as biological material In general biological evidence examinations precede any latent print examination Appropriate steps should be taken including consultation with the submitting party and or a latent prints examiner to ensure the appropriate course of action for the evidence presented In some cases it may be best if one type of examination is chosen over another If an analyst finds a distinct print during examination appropriate steps should be taken to preserve the print and inform the officer Storage of Evidence Biological evidence must be properly stored to preserve biochemicals assayed in body fluid identifications and DNA typing for current and future analyses Storage conditions for all types of evidence present must be considered so that the evidence is not compromis
79. these antibodies is immobilized at the test region on the membrane The upstream control region and the region of the internal standard between control and test region contain immobilized polyclonal goat anti mouse antibodies The amount of antibody at the internal standard is adjusted to a color intensity of the line which is equal to the color intensity of the test line at a PSA concentration of 4 ng ml A glass fiber pad downstream of the membrane is used for sample loading and transmission to a second fiber pad with the dried and gold labeled second monoclonal murine anti PSA antibody PSA at the sample will bind to the remobilized gold labeled antibody and form a PSA gold labeled anti PSA antibody complex Through the capillary effect of the membrane the reaction mixture including the complex mixture is carried upwards with the fluid In any case the colored gold labeled anti PSA antibody will bind to the anti mouse antibody at the control region and the region of the internal standard thus developing two red lines one at the control region and one at the region of the internal standard These two lines are independent of the existence of PSA in the sample and indicate only the correct execution of the test If the sample contains PSA the PSA gold labeled anti PSA antibody complex will bind to the immobilized monoclonal antibody of the test result region that recognizes another epitope on the PSA molecule sandwich complex The binding is
80. ts for precipitate If a precipitate has formed in either reagent ensure that the precipitate is back in solution before use Luminol mixture should be labeled with the appropriate lot number and expiration date The mixture will expire one hour after use 4 2 4 Phenolphthalein Test Kit Scope PHT Test Kit Solutions B and C are used for presumptive blood identification by oxidizing phenolphthalin colorless in a basic solution such as the test reagent to phenolphthalein pink in the presence of heme and hydrogen peroxide Instructions for use and interpretation are in the phenolphthalein test kit procedure Safety This reagent presents the following hazards FAD BIO SOP 4 2 Page 6 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Standard Operating Procedures Biology 4 Guality Assurance Solution B Flammable Handle with care Harmful or fatal if swallowed inhaled or absorbed through skin Avoid contact with eyes skin and clothing Light sensitive Avoid light heat sparks and flame Keep container tightly closed Solution C Handle with care May be harmful if swallowed inhaled or absorbed through skin Avoid contact with eyes skin and clothing Avoid heat sparks and flame Keep container tightly closed Wear gloves lab coat and mask during preparation and use Broken skin should be covered Equipment and Supplies e Solution B e Solution C e Sterile diH O e Steri
81. ts to thaw to room temperature Mix the 2X Master Mix and 20X Primer IPC Mix by brief vortexing 2 Make a set of positive standards as described in the Previous section entitled Preparing the DNA Quantification Standards a If standards exist and are less than two weeks old you may use them instead of making new standards 3 Prepare a Master Mix consisting of the following reagents and volumes a The Plexor HY Worksheet will include the necessary volumes for the master mix b The worksheet includes a correction factor for pipetting loss Volume Per Component Reaction HL Plexor HY 2X Master Mix 11 Plexor HY 20X Primer IPC Mix 1 1 Water Amplification Grade 7 7 4 Vortex the Master Mix for 3 to 5 seconds then briefly centrifuge the tube 5 Dispense 18 uL of the Master Mix into each reaction well a Make sure the plate is correctly oriented A1 will be to the top left FAD BIO SOP 14 1 Page 3 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification 6 Add 2 uL of each sample standard or control TE for negative control into the applicable wells 7 Seal the reaction plate with the adhesive cover 8 Centrifuge the plate at 3000 rpm for about 20 seconds in a plate centrifuge with plate holders to remove any bubbles 9 Load the plate into the plate holder of the 7500 Ensure that the plate is corre
82. verview The Biology Standard Operating Procedure SOP manual specifies procedures for routine examinations and analyses of biological evidence for human identification It is approved for use in the Biology section of the laboratory Within the scope of that purpose it is intended to ensure effective and efficient use of the laboratory facilities for the benefit of all user agencies It incorporates the quality assurance elements necessary to ensure the reliability and uniformity of analyses and reported conclusions Each approved revision of this manual will be version controlled and archived for retrieval by date of authority Only the approved revision in effect at the time of analysis governs the analysis The official version of this controlled document shall be published on the laboratory s intranet webpage and can be viewed from network computers Any deviation from accepted protocol requires approval as outlined in the Methods section of the quality manual and must be documented in the case file This SOP is only one part of the policies and procedures that govern all work performed by the Biology section The Biology section will follow the requirements detailed herein in addition to the following e Parent Agency General Orders Policy e Laboratory Quality Manual e Laboratory Safety Manual e Laboratory Biology Training Manual FAD BIO SOP 1 1 Page 2 of2 Issued by Technical Leader Revision Date May 2 2014 Effective Date June
83. vic M 1991 Detection of semen and blood stains using Polilight as a light source Forensic Science International 51 289 296 FAD BIO SOP 6 1 Page 6 of 6 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Forensic Biology Screening Semen Detection Forensic Analysis Division Standard Operating Procedures Biology 7 Semen Detection 7 Semen Detection Please refer to SOP 44 Reagent Quality Control for quality control testing storage labeling and expiration guidelines of the Acid Phosphatase Spot Test Alternate Light Source SERI Christmas Tree Stain Phosphate Buffered Saline 1X and SERATEC PSA Semiguant test devices Many samples submitted to the Crime Laboratory are tested for the presence of semen Generally a sample will be subjected to a presumptive acid phosphatase test followed by a confirmatory microscopic examination for the presence of spermatozoa If the sample is not microscopically positive a third test the test for the presence of prostate specific antigen may be warranted and executed 7 1 Alternate Light Source Scope The Alternate Light Source ALS lamps within this lab will be used for detection of forensic semen stains These lamps provide intense light of specific wavelengths through a hand held wand Some dried semen stains on cloth are detectable visually because their color off white or yellow is different from that of the material on which the semen h
84. vigate to the Desktop and select the Plexor HY template 4 Click Open or double click the template file NOTE The following box will appear any time you open close save or analyze the file Always click yes to navigate pass the box SDS v1 2 A Suggestion IF you include the SYBR Green dye then you may want to include a Dissociation stage Likewise if you include a Dissociation stage then you may want to include the SYBR Green dye Do you want to Analyze anyway 5 Click File gt Save As 6 Name the run with initials and date i e AB092012 7 Save the file to the Desktop 8 Click on the Instruments tab and click Start to start the run s4 7500 System SDS Software JLP092412 Abso a File view Tools Instrument Analysis Window Help OS h ek Ara rH we ETA Instrument Results Instrument Control LC smn Estimated Time Remaining hh mm 9 Ensure that the Estimated Time Remaining shows a time before leaving the room FAD BIO SOP 14 1 Page 7 of 12 Issued by Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 14 Plexor HY Guantification Analyzing run and Exporting files with 7500 SDS Software 1 When the run is complete the instrument will display a successful run completion message 2 Click okay 3 Omitting unused wells a Select the unused wells according to your worksheet on the Results tab b With the well selected hit Ctrl M c The omitted wells will
85. wers to ensure proper transcription of results by the author of the results from If performing quantification for proficiency samples the analyst should create his her own DNA standards Only one proficiency results form will be completed for proficiency tests to ensure that only the most complete form is submitted to the proficiency testing provider in order to be included in the provider s published external summary report The screening analyst shall complete the proficiency test results form if the test does not proceed to DNA analysis the DNA analyst shall complete the proficiency test results form if the test does proceed to DNA analysis FAD BIO SOP 4 2 Page 12 of 12 Issued by Technical Leader Revision Date August 26 2014 Effective Date September 1 2014 Forensic Biology Screening Evidence Evaluation and Handling Forensic Analysis Division Standard Operating Procedures Biology 5 Evidence Evaluation and Handling 5 Evidence Evaluation and Handling The Biology section provides body fluid identification semen and blood and performs the collection and preservation of trace evidence and possible contact DNA evidence 5 1 Case Acceptance and Evaluation Refer to the Quality Manual for procedures regarding the submission of evidence into the Crime Laboratory Before a case is worked the case and the reguested examinations will be evaluated The examiner should be aware of the reguested examinations and when possibl
86. y 1 cm of a stain may be consumed for this procedure 2 Add approximately 200 ul of 1X PBS to the sample More PBS may be required to ensure the sample is fully submerged 3 The extract s may be placed at room temperature in an ultrasonic cleaner for 5 15 minutes Alternatively the extracts may be soaked for 30 minutes 24 hours in refrigerated storage 4 Centrifuge the extract s briefly 5 The extract containing any cellular debris will be used for microscopic spermatozoa examination and may also be used for p30 testing 6 Store the extract refrigerated when necessary for up to 24 hours Beyond 24 hours the extract should be stored frozen Substrate controls may be included in body fluid identification tests as appropriate 7 4 Microscopic Spermatozoa Examination Scope Spermatozoa sperm detected on an evidence sample confirm the presence of semen the test is therefore a confirmatory test This procedure uses the SERI Christmas Tree Stain which is a Kernechtrot Picroindigocarmine differential biological stain that assists in scanning slides for the presence of spermatozoa The test consists of a two stain method which dyes the tip of sperm heads pink the bottom of sperm heads red the tails green or blue and any epithelium green with red nuclei Human sperm are flagellated with a total length of about 50 um The sperm cell head generally is oval flattened at the anterior end with dimensions about 4 6 um x 2 6 um x 1
87. y Technical Leader Revision Date May 2 2014 Effective Date June 1 2014 Standard Operating Procedures Biology 8 Trace Observation Collection Tape Lifting clear tape is pressed over the area potentially bearing trace evidence This application is repeated until all areas of interest are covered New tape should be used once the tape in current use loses its stickiness Tape s will be placed on the inside surface of a clean cut open zipper bag or a sheet protector The bag protector will be labeled as to its contents Retaining in butcher paper Trace evidence may also be preserved by retaining the evidence item in butcher paper In some instances however it may be most appropriate to repackage the item using the original packaging For example a swab with an embedded hair or fiber may be repackaged in the original swab carton The method of packaging selected should prevent damage deterioration and or loss of the trace evidence The evidence item may only be re opened in a clean trace environment to prevent contamination and or loss Place all collected trace evidence in the original evidence container with the item from which it was collected The observation or collection of trace evidence must be documented on the appropriate examination documentation Examiners should not make any attempt to identify on their worksheets what was collected i e hair fiber etc unless they also possess current training in the disciplines
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